Cytokines as therapy for opportunistic fungal infections

478

73rd FORUM

IN iMMUNOLOGY

Cytokines as therapy for opportunistic B.J. Kullberg

(‘)(*)

and E.J. Anaissie

fungal infections @)

“) Catholic University Nijmegen, Nijmegen (The Netherlands), and (2) The University of Arkansas for Medical Sciences, Little Rock, AR (USA)

Introduction Recently, great progress has been made in the development of antifungal therapy. New classes of antifungal drugs have been introduced, that bear promise for achieving cure from infection and a lower incidence of adverse effects (Uzun and Anaissie, 1996). Despite these developments, treatment failure is still a significant problem, amounting to 20-30% of patients with the most common opportunistic fungal infections (Anaissie et al., 1996a; Saag et al., 1992). In specific groups of patients, such as those with persistent neutropenia, failure rates are even substantially higher (Bodey, 1984; Denning, 1996). Resolution of these infections is often dependent on recovery from granulocytopenia or restoration of cellular immunity, indicating that host defence mechanisms are extremely important in the clearance of these pathogens. Therefore, immunotherapy aimed at enhancing host defence mechanisms may prove extremely productive in this field. Such interventions may be targeted at increasing the numbers of phagocytic cells, modulating the kinetics of these cells at the site of infection or activating phagocytes to kill the pathogenic fungal organisms more effectively. These effects of professional phagocytes are controlled by the differentiation of T helper cell subsets, and this Thl/Th2 balance itself may be the target of immunotherapy as well. For many decades, investigators have attempted to enhance the non-specific resistance to fungal infection by injection of microorganisms, such as Mycobacterium bovis BCG or Candida albicans (Van ‘t Wout et al., 1992; Vecchiarelli et al., 1989a), or microbial cell wall constituents, such as bacterial endotoxin or mummy1 dipeptides (Cummings et al., 1980; Tansho et al., 1994). Most of these compounds are known to stimulate the synthesis of proinflammatory cytokines interleukin-1 (ILl), interferon-y (IFNy) or tumour necrosis factor-cc (TNFa). The immunomodulatory effects of such

agents are at least partly mediated by the induction of these cytokines (Kullberg and Van ‘t Wout, 1994). The recent availability of a wide range of recombinant cytokines and haematopoietic growth factors has now opened the door to the application of immunotherapy in invasive fungal infections.

In vitro studies Neutrophils and mononuclear phagocytes (monocytes and macrophages) are of paramount importance in host defences against invasive mycoses. Hyphae of C. albicans and Aspergillusfumigatus are damaged and killed by neutrophils in vitro, and the hyphae of both fungi stimulate neutrophils to undergo a respiratory burst and to degrauulate (Diamond and Krzesicki, 1978 ; Diamond et at., 1978 ; Levitz and Farrell, 1990). Oxidative phagocyte products (e.g. hydrogen peroxide) and granule contents (e.g. defensins) are released after stimulation and can kill C. albicans and A. jirmigatus (Diamond aud Krzesicki, 1978; Lebrer et al., 1988). This is in agreement with the observation that patients with chronic granulomatous disease (CGD), a rare specific disorder of phagocytes which are defective in their ability to generate oxidative products, are prone to develop severe invasive aspergillosis and candidiasis. Treatment of neutrophils from CGD patients with recombinant IFNy (rIFNy) partially restores the oxidative defect, as well as their ability to kill A. fimigatus hyphae in vitro (Ezekowitz et al., 1990; Rex et al., 1991). In the normal host, a wide variety of effects of IFNT on neutrophils has been demonstrated, including stimulation of migration and adherence, phagocytosis and oxidative killing. Treatment of neutrophils with rIFNy has been shown to enhance their ability to damage and kill hyphae of C. aZbicansand A. fumigants in vitro (Djeu et al., 1986; Roilides et

ReceivedApril 9, 1998. (*) Correspondence to: Bart Jan Kullberg, Nijmegen,

The Netherlands.

Department

of Medicine

(541),

University

Hospital

Nijmegen,

PO Box

9101,

6500 HB

IMMUNITY al., 1993a) as well as the intracellular killing of C. albicans blastospores after phagocytosis (Kullberg and Van ‘t Wout, 1994). C. albicans and C. neoformans can directly bind to T and NK cells and stimulate IFNy gene expression and release, by apparently non-MHC-restricted mechanisms (Levi& and North, 1996).

There is abundant evidence that IFNy is the main cytokine involved in macrophage activation. The capacity of murine IFNy to activate macrophages in vitro to display enhanced fungicidal activity has been described for a large variety of fungal organisms, such as Blasromyces dermariridis (Brummer et al., 1988), Coccidioides immitis (Beaman, 1991), Paracoccidioides brasiliensis (Brummer et al., 1988), Hisroplasma capsularurn (Wu-Hsieh and Howard, 1992), C. neoformans (Perfect er al., 1987), C. albicans (Brummer et al., 1985 ; Vecchiarelli et al., 1989b) or A. fumigarus (Roilides er al., 1994). In addition, it has been found that stimulation of human venous endothelial cells with rIFNy in vitro leads to a significant reduction of the invasion of these cells by C. albicans (Ibrahim et al., 1993). This suggests that modulating the integrity of the vascular endothelial lining by cytokines may play a significant role in the susceptibility of the host to haematogenously disseminated yeasts. The effects of IFNy on cellular host defence mechanisms are closely linked to those of TNFcx. Activation of murine macrophages with IFNy leads to production of TNFol, which then serves as an intermediate for the intracellular killing of microorganisms (Langermans er al., 1992). Also, TNFa is induced after incubation of monocytes or macrophages by C. neoformans and C. albicans blastospores or C. albicans hyphae (Blasi et al., 1994; Jeremias et al., 1991; Levitz er al., 1994). Although the massive production of TNFa and its release into the circulation during severe sepsis are responsible for the development of shock, organ failure and subsequent death, small amounts of TNFcl appear to be essential for host resistance against infections. Incubation of human peripheral blood monocytes with rTNFcl inhibits the intracellular outgrowth of Coccidioides immiris (Beaman, 1991). TNFa has been demonstrated to enhance the phagocytosis of C. neofor-mans by murine macrophages in vitro (Collins and Bancroft, 1992), and neutralization of TNFa in vivo may also impair other host defence mechanisms to cryptococcosis, such as the formation of nitric oxide (Granger et al., 1988). TNFa causes neutrophilia, and increases neutrophil migration, adherence, degranulation, and respiratory burst (Steinbeck and Roth, 1989). Incubation of human neutrophils with rTNFa increased the intracellular killing of C. albicans (Djeu et al., 1986), whereas rTNFcl markedly impairs the capacity of neutrophils to kill C. albicans hyphae extracellularly (Diamond et al., 1991).

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Interleukin-1 shares many properties with TNFol. Divergent opinions exist about the capacity of IL1 to activate macrophages in vitro for killing of C. albicans or other microorganisms (Kullberg and Van ‘t Wout, 1994; Langermans et al., 1992; Vecchiarelli et al., 1989b). Significant differences have been found in the ability of macrophages obtained from different anatomical sites to be activated for killing of fungi. Also, differences in antifungal capacity between macrophages from different species have been noted. For example, the L-arginine-dependent generation of nitric oxide in macrophages is at least partly responsible for their intracellular antimicrobial activity in mice (Vazquez-Torres er al., 1995). This mechanism is primarily under the control of ILl, in a synergistic fashion with IFNy and TNFa (Billiau, 1996). The lack of nitric oxide generation in human monocytes may explain the absence of antifungal effects in these cells, whereas murine macrophages have displayed extensive antifungal activity, e.g. against Cryprococcus neoformans (Levitz and Ferrell, 1990). The cell-mediated immune response in mice is driven by distinct subsets of CD4+ T cells, termed Thl and Th2 cells. Thl cells are associated with the production of ILl, TNFa, IFNy, and IL12, as well as with protection against C. albicans infection. In contrast, a shift towards a Th2-type response is associated with production of IL4 and ILlO, and with progressive candidiasis. This in is agreement with the notion that incubation of macrophages with rILl0 inhibits the production of ILl, IL12 and TNFcx in vitro. Specifically, rILl0 induced dosedependent inhibition of ILlp and TNFol release by human monocytes stimulated by C. albicans or C. neoformans&evitz er al., 1996). Conversely, treatment of mice with specific monoclonal antibodies to either IL4 or IL10 enhances the ability of their macrophages to kill C. albicans in vitro (Romani et al., 1992, 199413). Apart from the relative absence of IL4 and IL10 production, the presence of at least three cytokines is crucial for the development of a protective Thl-type response : IL 12, IFNy, and transforming growth factor-p (TGFP) (Spaccapelo et al., 1995). Of these factors, IL12 is considered the most important determinant of Thl differentiation. During infection with C. albicans, IL12 appears to be produced not only by macrophages but also by neutrophils (Romani et aZ., 1997a). This underscores the role of neutrophils, not only as phagocytic cells but also as modulators of the immune response. Since IL12 has been shown to induce ILlO, the Thl-inducing effect of IL12 in candidiasis as well as in other types of infection may eventually be antagonized by the induction of IL10 (Romani et al., 1994a). The colony-stimulating factors (CSF) am able to augment the numbers of circulating phagocytes and

73rd FORUM their precursors in the bone marrow. Moreover, CSF have been shown to enhance activation of the fungitidal capacity of phagocytic cells in vitro. Incubation of macrophages with M-CSF enhances the killing of Candida and Cryptococcus species (Brummer and Stevens, 1994; Karbassi et al., 1987). Neutrophil function is enhanced by rG-CSF and rGMCSF, and incubation of neutrophils with either of these growth factors leads to increased killing of Ctyptococcus, Candida or Aspergilius species in vitro (Levitz, 1991; Natarajan et al., 1997; Yamamoto et aZ., 1993). It appears that rG-CSF-activated neutrophils exert fungicidal activity particularly against pseudohyphal elements of C. albicans (Kullberg et al, 1998; Roilides et al., 1995), but less against Candidu blastoconidia (Roilides et al., 1993a). In an experimental model in vitro, the ability of neutrophils to damage Aspergillus hyphae can be abrogated by incubating the cells with corticosteroids, underscoring that steroids are a risk factor for invasive aspergillosis (Roilides et al., 1993b). Incubating neutrophils with rG-CSF effectively reverses the deleterious effect of steroids in this model, reinstating their capacity to kill AspergiZlus in vitro (Roilides et al., 1993b). Incubating neutrophils with rIFNy or the combination of rG-CSF and rIFNy has similar effects (Roilides et al., 1993b). These in vitro effects are consistent with the ability of rG-CSF administered in vivo to enhance the activity of neutrophils obtained from normal human volunteers against opportunistic fungal pathogens (Liles et al., 1997). rG-CSF significantly enhanced the neutrophil-mediated killing of A. fumigatus and Rhizopus arrhizus. In addition, rG-CSF administration primes neutrophils for increased oxygen radical production in response to extracts of Candida, Aspergillus and Rhizopus organisms (Liles et al., 1997). Likewise, when administered to AIDS patients, rG-CSF effectively restores the capacity of their peripheral blood neutrophils to kill C. albicans and C. neoformans to normal levels (Vecchiarelli et al., 1995). During the process of haematogenous dissemination, the invading fungi adhere to and penetrate the endothelial lining of the blood vessel to invade the deep tissues. It has been found that C. albicans is actively phagocytized by endothelial cells during this process of tissue invasion, and that Candida stimulates endotbelial cells to synthesize a variety of adhesion molecules, eicosanoids and cytokines, including ILl, IL8 and TNFol (Filler et al., 1996; Rotrosen et aZ., 1985). These responses likely mediate the recruitment of neutrophils to the area of infection as well as their activation for antifungal activity. This assumption is in agreement with the finding that both neutrophils and monocytes exhibit a significantly enhanced anticryptococcal activity in the presence of endothelial cells, and that IL1 and IL8 secreted by endothelial cells are able to activate

IN IMMUNOLOGY neutrophils for killing of C. neofomans (Roseff and Levitz, 1993). Therefore, it is assumed that endothelial cells have a crucial role in determining the magnitude and profile of the host inflammatory response to fungal infections. In vziw animal studies In spite of the differences in antifungal capacity of phagocytes from different species, as well as the interspecies differences in cytokine patterns in response to infection, animal models have been essential to study the potential effects of immunotherapy with recombinant cytokines for invasive mycoses. Administering recombinant IFNy has a beneficial effect on the course of lethal disseminated cryptococcosis in rats (Joly et al., 1994). Conversely, neutralization of either IFNy or TNFol by specific monoclonal antibodies impairs the resistance of mice to cerebral cryptococcosis, and simultaneous administration of both antibodies further exacerbates infection (Aguirre et al., 1995). In a model of disseminated aspergillosis in mice, combined administration of rIFNy and rTNFol reduced the mortality and organ burden of Aspergillus (Nagai et al., 1995). Administration of rIFNy alone has a beneficial effect on the course of experimental candidiasis in mice, even when the infection had already been established for several days (Kullberg et al., 1993). In mice with impaired host defence due to hydrocortisone pretreatment, rIFNy also reduced the outgrowth of C. albicans in the organs. In contrast, the beneficial effect of rIFNy on disseminated candidiasis could be abolished by pretreatment of mice with cyclophosphamide, suggesting that the observed effect of rIFNy was mediated by activation of neutrophils. This is also supported by the observation of enhanced killing of C. albicans by peritoneal and peripheral blood neutrophils from rIFNytreated mice (Kullberg et al., 1993). In line with the requirement of TNFcx for antifungal activation of phagocytes, treatment of mice with monoclonal antibodies against TNFol enhances the mortality to experimental systemic cryptococcosis and leads to increased numbers of yeasts in the brain and meninges as compared to control mice (Aguirre et al., 1995 ; Collins and Bancroft, 1992). Likewise, treatment with either anti-TNFol or pharmacological inhibitors of TNFa production leads to enhanced mortality and increased outgrowth of C. albicuns during disseminated candidiasis in mice (Louie et al., 1994; Netea et al., 1995; Steinshamn and Waage, 1992). The effect of anti-TNFa is not seen in neutropenic animals, supporting the in vitro observations indicating that TNFcl is required to activate PMNs for killing bf Cundidu and that its inhibition restricts the intracellular killing of the yeasts.

IMMUNITY A single injection of either rILlcl or rILlP has been shown to protect neutropenic mice from lethal disseminated candidiasis, and significantly decreases the numbers of C. albicans in the kidneys and spleen of infected normal mice and of mice rendered immunocompromised by cyclophosphamide, hydrocortisone acetate or total body irradiation (Kullberg et al., 1992; Kullberg et al., 1990). Although rIL1 has been known to enhance the production of granulocytes, their release from the bone marrow, and subsequently their migration to the site of infection (Kullberg et al., 1990), its protective effect in disseminated candid&is is independent of the presence of neutrophils. Even during complete agranulocytosis after total body irradiation, the outgrowth of C. albicans was reduced by ILl. Histologic examination of the organs of mice infected with C. albicans did not show any differences in the numbers of granulocytes at the foci of infection between ILltreated mice and controls (Kullberg et al., 1990). Moreover, IL1 did not increase the number of granulocytes in the peripheral blood or at the site of infection in mice with a C. albicans peritonitis (Kullberg ef al., 1991). Modulation of cytokine production, cytokine receptor expression and induction of acute phase proteins and other humoral factors are thought to contribute to the protective effect of rIL1 in these models (Vogels et al., 1994; Vogels and Van der Meer, 1992). In systemic candidiasis as well as in invasive aspergillosis or cryptococcosis in mice, the development of protection is associated with a Thl cytokine profile, whereas the presence of Th2-type cytokines has been linked to the development of chronic disease (Cenci et al., 1997; Mencacci et al., 1995; Murphy, 1993; Romani et al., 1994b). Failure to clear a subacute or chronic infection with C. albicans corresponds to sustained production of IL4 and IL10 in susceptible mouse strains, whereas so-called constitutively resistant strains produce significantly less IL4 and IL10 (Romani et al., 1994b). Neutralization of either of these cytokines by specific monoclonal antibodies augments host resistance, leads to increased survival of infected mice and enhances the ability of their macrophages to kill C. albicans in vitro (Romani et al., 1992, 1994b). In a different approach, recombinant soluble receptors to IL4, which bind and neutralize circulating IL4, were able to cure potentially lethal subacute disseminated candidiasis, inducing a Thl-type response with high levels of IFNy (Puccetti et al., 1994). Likewise, impaired neutrophil activity against A. fumigatus hyphae, observed in susceptible mice, was concomitant with a predominant production of IL4, and treatment with soluble IL4 receptors led to cure from the infection (Cenci et al., 1997). Administration of rIL12, a strong stimulator of a Thl-type response, reduced the severity of dissemi-

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nated cryptococcosis in rats, and the combination of IL12 and fluconazole had an additive effect (Clemons et al., 1994). In the liver, fluconazole completely failed to contain the infection, whereas treatment with rIL12 was effective in reducing the outgrowth of C. neoformans (Clemons et al., 1994). The outcome of rIL12 treatment in experimental disseminated candidiasis has been divergent. Whereas administration of rIL12 is associated with recovery from subacute Candida infection, and IL12-neutralizing antibodies impaired a protective Thl response, it appears that rIL12 exacerbates the course of an acute lethal C. albicans infection in mice (Romani et al., 1995, 1997b, 1994a). Administration of monoclonal antibodies to IFNy reverses the deleterious effects of rIL12 (Romani et al., 1995), and it may be hypothesized that excess rIL12 during the acute phase of Candida sepsis leads to extensive IFNy production, inducing organ failure and death Various animal studies suggest a beneficial effect of rG-CSF on experimental infection with C. albicans (Uchida et al., 1992), A.fwnigatus (PolakWyss, 1991; Uchida et al., 1992) and C. neoformans (Uchida et al., 1992) in neutropenic animals, and neutrophil recovery rather than specific antifungal activation of phagocytes is thought to be responsible for these effects. In non-neutropenic animals, administration of a single dose of murine rG-CSF reduced the mortality and significantly decreased the outgrowth of C. albicans in the organs of the animals (Kullberg et al., 1998). Although the low dose of rG-CSF that was administered in that study had no effect on the numbers of neutrophils in the circulation during acute disseminated candidiasis, the histopathology of the kidneys showed a noticeable increase in the numbers of neutrophils at the actual sites of infection, indicating that neutrophils that were mobilized by rG-CSF were rapidly directed to the infected organs. Outgrowth of hyphal forms of C. albicans was almost completely prevented in rG-CSFtreated animals. Treatment with rG-CSF not only increased the influx of neutrophils to the site of infection, but also augmented the production of reactive oxygen intermediates by these cells (Kullberg e? al., 1998). Remarkably, treatment with rGCSF leads to a reduction in the circulating concentrations of TNFa and ILla during the infection, in spite of the increased influx of inflammatory cells at the sites of infection. An additive effect of rGCSF and antifungal drugs has been demonstrated in several non-neutropenic mouse models (Graybill er al., 1995; Herbrecht et al., 1996). Treatment with rM-CSF has been associated with a favorable outcome in models of subacute or chronic disseminated candidiasis (Cenci et al., 1991; Vitt et al., 1994), but others found that administration of rMCSF aggravated the course of experimental candidemia in mice (Hume and De&ins, 1992).

482 Preventive

73rd FORUM use of cytokines

in patients

A beneficial effect of IFNy strated in patients with CGD. In randomized, placebo-controlled of invasive fungal infections in patients who received rIFNy reduced from 24 % to 4% in 2 199 1).

has been demona large, prospective, trial, the incidence the group of CGDwas significantly years (GaIlin et al.,

In cancer patients, cytokines have been used both to shorten the duration of chemotherapy-induced granulocytopenia (Anaissie et al., 1996b; Antman et al., 1988; Crawford et al., 1991; Gerhartz et al., 1993 ; Pettengell et al., 1992) and as adjunctive therapy in patients with febrile neutropenia (Biesma et ab, 1990; Maher et al., 1994). However, their role in the prevention of fungal infections has not been established in these studies. The potential beneficial role of rGM-CSF has been evaluated in a recent prospective, randomized, placebo-controlled, phase III study of 124 patients aged between 55 and 70 years with acute myelogenous leukemia (Rowe et al., 1995, 1996). Patients were randomized to receive either placebo or rGM-CSF from day 11 postinduction until PMN recovery. Patients who achieved complete remission received the same medication (rGM-CSF or placebo) as the consolidation therapy they had for induction therapy. rGMCSF was associated with a higher rate of complete response than placebo, longer overall survival (median of 10.6 months in the rGM-CSF group vs 4.8 months in the placebo group (P=O.O48)) and lower infection-related toxicity (P=O.O15). The mortality in patients with pneumonia and who were randomized to rGM-CSF was 14% (2/14 patients), compared with 54% (703 patients) for those in the placebo group (P=O.O46). Furthermore, the fungal infection-related mortality rate was only 2 % for those randomized to receive rGM-CSF compared with 19% for those receiving placebo (P=O.O06). Among rGM-CSF recipients, three of four with aspergillosis and all three patients with candidiasis survived, compared with two of seven and one of four in the placebo group, respectively. Significant morbidity and mortality from invasive fungal infections occur in patients with apparently adequate neutrophil counts. Cytokines, particularly IFNy, GM-CSF, and G-CSF, increase the number and/or stimulate the function of phagocytic cells. It is tempting to speculate that the use of these cytokines as prophylactic agents in this setting might be valuable. More specifically, rGM-CSF or rGCSF, alone or in combination with IFNy, could be used immediately after engraftment following allogeneic transplantation or even autologous transplantation in certain settings. These agents could thus be used to stimulate phagocytic cells and hence decrease the morbidity and mortality from aspergil-

IN IMI4UNOLOGY losis, which has become the leading cause of death in these patients. A prospective randomized trial evaluating the role of I-GM-CSF after engraftment is currently in preparation. Cytokine therapy fungal infections

in patients

with

established

The first randomized multicentre clinical study addressing cytokine therapy for invasive mycoses is currently underway in Europe and compares fluconazole alone versus IG-CSF with fluconazole in nonneutropenic patients with disseminated candidiasis. Preliminary analysis of the blinded data shows that increasing the numbers of circulating neutrophils during severe candidiasis strongly correlates with accelerated clearance of Candida from the bloodstream and reduced mortality, even in this non-neutropenic study population (Kullberg et al., unpublished data). Three case reports have been published describing successful therapy with antifungal agents in combination with rG-CSF in one patient each with invasive mucormycosis (Gonzalez et al., 1997) or fusariosis (Hennequin et al., 1994), and haematogenous infection with Trichosporon spp. (Grauer et al., 1994). A patient with CGD and Paecilomyces varioti infection was treated with antifungal chemotherapy and rIFNy (Williamson et al., 1992). Disseminated infection with P. boydii in a patient with CGD was reported to be cured with a combination of amphotericin B and rIFNy (Phillips et al., 1991). Moreover, therapy with rIFNy and rG-CSF substantially improved PMN respiratory burst and was associated with clinical improvement of a patient with leukaemia and disseminated aspergillosis (Dignani, unpublished data). Recently, two patients with progressive chronic disseminated candidiasis have been described, who responded to rIFNy therapy, which had initially been combined with rGM-CSF (Poynton et al., 1998). It is suggested that rIFNy in particular was responsible for the observed effect, rather than the rGM-CSF. This is in agreement with the results of others, who found no beneficial effect of rGM-CSF in an open study of 17 patients, 8 of whom had candidaemia, 8 had pulmonary aspergillosis, and 1 had fusariosis. Of these patients, 4 developed severe capillary leak syndrome and 8 failed to show bone marrow recovery, leading to death due to fungal infection in 6 cases (Maertens et al., 1997). In an earlier pilot trial, eight patients with documented refractory fungal infections were treated with glycosylated (E. coli) GM-CSF plus amphotericin B (Bodey et al., 1993). Four of five patients with candid&is, one of two with aspergillosis, and the one patient with trichosporonosis improved on this therapy. GM-CSF was used at a relatively high dose in this study (400 mg/m’/d) and

IMMUNITY

was associated with capillary leak syndrome. However, given the limited nature of the study and the lack of a comparison group, no conclusions about actual efficacy can be drawn. In addition, a patient with Fusarium infection was treated with antifungal therapy, PMN transfusions and rGM-CSF (Spielberger et al., 1993). Patients who received rM-CSF showed enhanced monocyte function, including oxidative burst as well as antifungal activity by monocytes against C. albicans blastoconidia and A. fumigatus hyphae (Khwaja et al., 1991 ; Nemunaitis et al., 1991, 1993). A series of cancer patients were given MCSF combined with an antifungal agent as therapy for an invasive fungal infection (30 patients had Candida infections, 15 had Aspergillus and one had a Mucor infection) (Nemunaitis et al., 1993). Greater survival was observed in patients with Candidu infections and Kamofsky score >20 %, compared with historical controls. Unfortunately, this non-randomized study provides little data on the effect of the cytokine on the course of the fungal infection. White blood cell transfusions invasive fungal infections

in patients

with

In the past, several clinical trials on white blood cell transfusions (WBCTx) have been conducted in patients with neutropenia-related infections. In these studies, most patients had refractory bacterial infections, and they were randomized to receive treatment with WBCTx plus antibiotics versus antibiotics alone. Five of the studies showed that WBCTx could be life-saving among those patients with prolonged neutropenia (Alavi et al, 1977 ; Freireich et al., 1964; Graw et al., 1972; Herzig et al., 1977; Vogler and Winton, 1977), or could improve overall survival (Higby et al., 1975b), whereas two showed no benefit of WBCTx therapy (Fortuny et al., 1975; Winston et al., 1982). Unfortunately, none of these trials evaluated the role of this modality in patients with invasive fungal infections. In addition, this modality was abandoned because of the costs and toxicity to recipients including: fever, chills, hypotension, pulmonary infiltrates, respiratory distress (Andrews et al., 1976; Dana et al., 1981; Karp et al., 1982; Ward, 1970; Wright et al., 1981), transmission of cytomegalovirus (Hersman et al., 1982; Winston et al., 1980), graft-versus-host disease (Cohen et al, 1979; Weiden et al., 1981), alloimmunization (Ford et al., 1982; Schiffer et al., 1979) and haemolytic reactions (Chipping et al., 1980). Some authors also reported severe pulmonary toxicity with the simultaneous administration of amphotericin B and WBCTx (Wright et al., 1981). This toxicity has not been reported by others, suggesting that other variables such as infection and alloimmunization

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483

were responsible for pulmonary decompensation related to WBCTx therapy (Dana et al., 1981 ; DeGregorio et al., 1981; Dutcher et al., 1989). Three clinical studies (Aisner et al., 1978; Freireich et al., 1964; Higby et al., 1976) have demonstrated that the higher the number of cells transfused per m* body surface area, the better the clinical response to WBCTx. Thus, dexamethasone became the drug of choice to stimulate the donor and generate a high number of cells for collection and transfusion (mean PMN yield 2.5 x lOlo) (Higby et al., 1975a). This approach never produced the very high yields thought desirable for consistent success (Freireich et al., 1964). Recently, the possibility was raised that administration of rG-CSF to WBC donors would increase their ANC to levels that would lead to a higher yield of better quality PMNs. In 1992, we treated 10 profoundly neutropenic patients with refractory fungal infections with daily WBCTx obtained from donors daily treated with 5 pg/kg of rG-CSF subcutaneously (Feldman et al., 1993). Half of the recipients showed a favourable outcome of the infection after WBCTx. Donors achieved a 4- to lofold increase of their ANC, and the mean PMN yield was 3.7~10”. Recipients achieved a median l- and 24-h post-transfusion ANC of 780 and 426/ul, respectively. In 1993, Bensinger et al. treated 8 more donors with the same dosage of t-G-CSF and obtained comparable PMN yields (mean 4.1 x lOlo) and 24-h post-transfusion ANC (median 570&l), but no data about clinical efficacy of WBCTx were provided (Bensinger et al., 1993). In a more recent study, we used rG-CSF-elicited WBCTx to treat 15 adult cancer patients with neutropenia (ANC c 5OO/ul) and documented and refractory fungal infections (Dignani et al., 1997). All patients had haematological malignancies and 7 had been treated with bone marrow transplantation. Infections included aspergillosis (7 patients), fusariosis (3), candidiasis (3) and trichosporonosis and unidentified mould infections (1 each). Appropriate antifungal therapy had failed to produce a response (median of 10 days prior to WBCTx) in these patients, who had been neutropenic for a median of 23 days. At the end of WBCTx therapy, 11 of 15 patients had a favourable response (9 improved and 2 stabilized), and in 7 of them the favourable response appeared to be substantially due to WBCTx. Those patients in whom WBCTx therapy was started early during neutropenia and shortly after diagnosis of fungal infection were more likely to respond. The favourable responses were still seen 3 weeks after the end of WBCTx therapy in 8 of 11 responders. This small pilot study demonstrated that rG-CSF-enhanced WBCTx therapy may be life-saving for patients with refractory neutropenia-related fungal infections and is safe to deliver.

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73rd FORUM

IN IMMUNOLOGY

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